I. RORgamma: The retinoid-related orphan receptor g (RORg) is a member of the nuclear receptor superfamily. To identify the physiological functions of RORg, mice deficient in RORg function were generated by targeted disruption. RORg -/- mice lack peripheral and mesenteric lymph nodes and Peyer?s patches indicating that RORg expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, while the number of circulating B cells is normal. The thymus of RORg-/- mice contains 74.4+ 8.9% fewer thymocytes than that of wt mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. TUNEL staining demonstrated a four-fold increase in apoptotic cells in the cortex of the thymus of RORg-/- mice. This was supported by the observed increase in annexin V-positive cells. RORg-/- thymocytes placed in culture exhibit a dramatic increase in the rate of "spontaneous" apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may at least in part be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a six-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORg-/- mice. Our observations indicate that RORg is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Recently, we found that RORg-/- mice are highly susceptible to the development of T-cells lymphomas. Altered expression of ROR leads to deregulation of the differentiation of thymocytes, increased proliferation of double negative thymocytes, which in turn increases the frequency of tumor formation. Recent studies have demonstrated that ROR receptors play a regulatory role in various inflammatory respeonses. RORa mice are more susceptible to endotoxin-induced inflammation. II. TAK1: The nuclear orphan receptor TAK1 functions as a positive as well as a negative regulator of transcription; however little is know about factors mediating its activity. Yeast two-hybrid analysis using the ligand binding domain of TAK1 as bait identified a novel TAK1-interacting protein, referred to as TIP27. TIP27 is a 27 kD nuclear protein that contains two zinc finger motifs. Confocal microscopy using TIP27 and several deletion mutants showed that TIP27 localized to the nucleus and that the carboxyl terminus containing a putative nuclear localization signal is important for its nuclear localization. TIP27 mRNA is expressed in several adult tissues but is most highly expressed in testis where it is induced at a specific stage of spermatogenesis. Mammalian two-hybrid analysis showed that TIP27 interacts specifically with TAK1 and not with several other nuclear receptors tested. Deletion mutation analysis determined that the region between Asp39 and Lys79 of TIP27, referred to as TAK1-interaction domain (TID), is critical for its interaction with TAK1. Moreover, it demonstrated that the TAK1-LBD from H3 till the carboxyl terminus is required for optimal interaction with TIP27. Pull-down assays demonstrated that TIP27 physically interacts with TAK1 and supported the importance of the TID. TIP27 is a strong repressor of DR1-dependent transcriptional activation by TAK1. This repression does not involve inhibition of TAK1 homodimerization or DR1 binding but appears to due to an inhibition of the recruitment of co-activators. Our studies indicate that TIP27 is an effective repressor of transcriptional activation by TAK1 and, therefore, may play a critical role in the regulation of several physiological functions by TAK1. Public Health or Environmental Health Significance: Nuclear receptors constitute a superfamily of ligand-dependent transcription factors, that include receptors for steroid hormones, retinoids, thyroid hormone, and eicosanoid metabolites, and orphan receptors for which ligands have no yet been identified. Nuclear receptors have been demonstrated to have critical regulatory functions in embryonic development and many other physiological processes. In addition, nuclear receptors have been implicated in a number of diseases, including cancer, diabetes, osteoporosis, and atherosclerosis. Since nuclear receptors function as ligand-dependent transcription factors, they can serve as targets for synthetic agonists and antagonists and provide a pharmacological tool to interfere into human diseases. Synthetic agonists for a number of nuclear receptors are being used in therapy for a number of diseases, including cancer, inflammation, and diabetes. In addition to natural ligands, xenobiotics can function as ligands for nuclear receptors, thereby providing a mechanism by which they influence physiological processes. Inversely, nuclear receptors have been shown to affect the metabolism of drugs and environmental chemicals by regulating the transcription of genes encoding a variety of metabolic enzymes.